Raffinose d galactose assay kit

Plasma raffinose concentration was determined using raffinose/D-galactose assay kit (Megazyme, Ireland) following manufacturer's instructions with modifications for use in a microplate. Briefly, 20 μl plasma samples were mixed with either 10 μl α-galactosidase (pH 4.6) or 10 μl distilled water for determining concentration of raffinose + free D-galactose or free D-galactose only, respectively, and mixtures were incubated at 25°C for 20 min. Distilled water was used as blank control. Next, 20 μl buffer containing sodium azide, 200 μl distilled water, and 10 μl NAD⁺ solution, were added, mixed, and absorbances read at 340 nm (A1 read) after 3 min. Then, 2 μl D-galactose dehydrogenase plus galactose mutarotase suspension was added, mixed, incubated at 40°C for 20 min, and absorbances read at 340 nm at the end of the reaction (A2 read). Raffinose concentration was calculated by:
V = final volume [ml]
MW = molecular weight of the substance (504.5 for raffinose) assayed [g/mol]
ε = extinction coefficient of NADH at 340 nm = 6300 [l × mol⁻¹ × cm⁻¹]
d = light path [cm] = 1
v = sample volume [ml] = 0.02
The 96 well plate was read twice with a Synergy HT multimode microplate reader (BioTek, Winooski, VT). Average concentration of individual samples was used with standard error of mean (SE).

In order to evaluate the resorptive capacity of the intestine, 30 mM of lactose were supplied with the luminal perfusion buffer and vascular galactose (derived from the luminal lactose) was determined by a commercially available assay kit (Raffinose/D-Galactose Assay Kit, Megazyme, Bray, Ireland). Due to initially high variations in galactose uptake during the equilibration phase, statistical analyses were only performed with samples from time points 60 min and beyond.